hDlg is the human homologue of the Drosophila discs large tumor suppressor. We have recently identified a novel protein termed GAKlN (Guanylate kinase Associated Kinesin) that binds to the guanylate kinase-like domain of hDlg and PSD-95. By sequence homology, GAKIN is a member of the kinesin superfamily of motor proteins. The aim of this proposal is to test the hypothesis that the GAKIN-hDlg interaction lays the foundation for a general paradigm of the coupling of MAGUKS to the microtubule-based cytoskeleton, and that this interaction is functionally important for the intracellular trafficking pathways. To test this hypothesis, we will examine: (1) Generality and specificity of hDlg interaction with GAKIN. We propose to map the GAKIN and hDlg binding sites using in vitro mutagenesis, protein expression, and BlAcore assays. We will determine the proximity of GAKIN binding and test for the existence of similar interactions within other members of the MAGUK family. Together, these studies are likely to elucidate a novel mechanism for the physical coupling of MAGUKs to microtubules via kinesin motors. (2) Identification of proteins associated with the hDlg-GA KIN complex. We will use large-scale protein purification and microsequencing techniques to identify the proteins associated with the hDlg-GAKIN protein complex. Identification of these components will be essential for complete understanding of scaffolding and transport functions of MAGUKs. (3) Functional characterization of the hDlg-GAKIN protein complex. We propose that the membrane trafficking and targeting of hDlg is mediated by GAKIN. To test this hypothesis, we will introduce specific mutants of hDlg and GAKIN that are functionally disabled for binding into lymphoid, epithelial, and neuronal cells and examine their effects on hDlg localization and receptor clustering. These studies are designed to test for the function of MAGUK-Motor link in the protein and membrane vesicle targeting of other MAGUKS. (4) Murine models of Dig deficiency. A prerequisite for testing the functional role of hDlg is the availability of cell lines lacking the endogenous hDlg or expressing a mutant form of hDlg lacking its guanylate kinase domain (Gukiess DIg). Using gene targeting techniques, we will generate a Gukless mutant in the murine ortholog of hDlg (mDlg) in mice. This aim will allow us to assess the physiological consequences of mDlg null mutation on the growth and proliferation of hematopoietic and neural cell types and whether mDlg acts as a tumor suppressor as observed for Dlg in Drosophila.